As a method of producing an optically active amino acid from an amino acid amide, for example, a method comprising utilizing a microorganism which contains an enzyme stereoselectively hydrolyzing an amide bond of a racemic amino acid amide cell processed product of such a microorganism is known as an industrially advantageous method (Patent Documents 1 and 2). Such an enzyme, which is a catalyst derived from a living body, has excellent characteristics that it allows a product of interest to be produced with extremely high reaction specificity under a moderate reaction condition. However, a kind of substrate to be catalyzed is limited, and a large amount of cells are required for reaction because of small amount of the enzyme to be produced by cells, and therefore, there is a technical problem that the method cannot be employed because of its economical disadvantage.
Therefore, in order to increase production amount of an enzyme having the above-mentioned activity, it has been attempted to prepare a transformant by cloning a DNA encoding an enzyme protein of an enzyme which stereoselectively hydrolyzes an amino acid amide by a gene recombination technique and then to produce an optically active amino acid from a racemic amino acid amide by using the transformant (Patent Documents 3 to 8 and Non-patent Document 1).
Patent Document 3, for example, discloses D-amidase derived from Variovorax, a DNA encoding the D-amidase, a plasmid, a vector and a microorganism each containing such a nucleic acid, a nucleic acid which hybridizes with the nucleic acid, primers for producing the nucleic acid, and the like. Patent Document 4 discloses a modified amino acid amidase and a method of producing D-amino acid using the same. However, these methods work only for D-amino acid amide, so the L-amino acid amides cannot be hydrolyzed by the methods.
On the other hand, as an example of an enzyme which acts on an L-amino acid amide, Patent Document 5 discloses an enzyme which stereoselectively hydrolyzes an amide bond of an L-amino acid amide and is derived from Pseudomonas azotoformans, and a DNA encoding the enzyme. The enzyme has a high activity to L-proline amide but has a low activity to other L-amino acid amides.
In addition, Patent Document 6 discloses an enzyme protein derived from Comamonas acidovorans and a DNA encoding the protein, and a method of producing an optically active organic acid using the enzyme protein. Patent Document 6 also discloses asymmetric hydrolysis of an amino acid amide in which only leucine amide and phenylalanine amide are used as a substrate. Further, Patent Document 7 discloses a protein having an amidase activity to stereoselectively hydrolyze α-amino acid amide and α-hydroxylic acid amide and a DNA encoding the protein. However, only t-leucine amide is disclosed as a preferable amino acid amide substrate. Furthermore, Patent Document 8 discloses a method of obtaining a microorganism containing a peptide amidase, a microorganism obtained by the method, a peptide amidase contained in the microorganism, and a method of using them. This enzyme has a high activity to dipeptide amide, N-acetyl amino acid amide, and protected amino acid amide, while it has no activity to hydrolyze an unprotected amino acid amide. Still further, Nonpatent Document 1 discloses an enzyme which stereoselectively hydrolyzes an amino acid amide having an alkyl group at position α. However, examples disclosed therein are limited to amino acids having a side chain of hydrocarbon type such as methylvaline, methylleucine, and methylphenylalanine, and there is no description at all about 2-alkylcysteine which has a mercapto group and an alkyl group in side chain.
The 2-alkylcysteine is a derivative of alkylcysteine which is an atypical amino acid other than typical natural amino acids, and has an alkyl group on the carbon atom at position α and multiple reactive substituents including mercapto group, amino group and carboxyl group in its molecule. In particular, optically active form of 2-alkylcysteine is a compound expected to be widely used as a production raw material for various industrial chemicals, pharmaceuticals, agricultural chemicals and the like, or a general chiral building block, and is also extremely useful from the industrial viewpoint, so a production method thereof which is industrially advantageous and inexpensive has been desired to be developed.
However, as described above, there has not been known an enzyme which has an activity to stereoselectively hydrolyze an amide bond in the L-2-alkylcysteine amide, and therefore, there has not been known a method of producing an optically active 2-alkylcysteine or an optically active 2-alkylcysteine amide by using such an enzyme. Further, there have been demands for an L-amino acid amide hydrolase which is highly active and highly stereoselective and has a wide substrate spectrum for other L-amino acid amides so as to be applicable to various amino acid amides useful as industrial raw materials, and methods of efficiently producing and using the enzyme.
Patent Document 1: JP-A-61-293394
Patent Document 2: JP-A-62-55097
Patent Document 3: JP-A-2003-225094
Patent Document 4: JP-A-2002-253256
Patent Document 5: JP-A-2003-250558
Patent Document 6: JP-A-08-256771
Patent Document 7: WO 00/63354
Patent Document 8: Japanese Patent No. 3112090
Non-patent Document 1: H. F. M. Hermes et al., Applied and Environmental Microbiology, Vol. 60, No. 1, p. 153-159, 1994